Wilms' tumour 1‐associating protein inhibits endothelial cell angiogenesis by m6A‐dependent epigenetic silencing of desmoplakin in brain arteriovenous malformation

Abstract Brain arteriovenous malformations (AVMs) are congenital vascular abnormality in which arteries and veins connect directly without an intervening capillary bed. So far, the pathogenesis of brain AVMs remains unclear. Here, we found that Wilms' tumour 1‐associating protein (WTAP), which has been identified as a key subunit of the m6A methyltransferase complex, was down‐regulated in brain AVM lesions. Furthermore, the lack of WTAP could inhibit endothelial cell angiogenesis in vitro. In order to screen for downstream targets of WTAP, we performed RNA transcriptome sequencing (RNA‐seq) and Methylated RNA Immunoprecipitation Sequencing technology (MeRIP‐seq) using WTAP‐deficient and control endothelial cells. Finally, we determined that WTAP regulated Desmoplakin (DSP) expression through m6A modification, thereby affecting angiogenesis of endothelial cells. In addition, an increase in Wilms' tumour 1 (WT1) activity caused by WTAP deficiency resulted in substantial degradation of β‐catenin, which might also inhibit angiogenesis of endothelial cells. Collectively, our findings revealed the critical function of WTAP in angiogenesis and laid a solid foundation for the elucidation of the pathogenesis of brain AVMs.

ischaemic attack are the most common relevant symptoms, 5 seriously endangering human health and life. But until now, the pathogenesis of brain AVMs is still unclear.

Desmoplakin (DSP) is a critical component of desmosome and
plays a central role in maintaining the structure and stability of the desmosome. 37 In addition, previous studies have demonstrated that DSP is essential for the mechanical integrity of epithelium and myocardium. [38][39][40] DSP gene mutations are associated with cutaneous or cardiac defects, such as palmoplantar keratoderma, skin fragility-woolly hair syndrome, lethal acantholytic epidermolysis bullosa, arrhythmogenic right ventricular cardiomyopathy and Carvajal syndrome. 41,42 In addition to cell adhesion, DSP has recently been found to be involved in other cellular processes such as proliferation, differentiation and carcinogenesis. 40,43 Surprisingly, DSP is not only related to epidermal integrity and cardiac function, but also to vascular development. For instance, ablation of DSP results in leaky and/or poorly formed capillaries, limiting embryonic development. 44,45 Therefore, it is of great significance to explore the relationship between DSP and the pathogenesis of brain AVM.
In the current study, we found that WTAP was down-regulated in brain AVM lesions compared with normal cerebral vessels, and knockdown of WTAP significantly inhibited tube formation of the human endothelial cells. To thoroughly investigate the specific molecular mechanism, we performed RNA transcriptome sequencing (RNA-seq) and Methylated RNA Immunoprecipitation Sequencing technology (MeRIP-seq) to screen downstream targets of WTAP.
Finally, we determined that DSP was stabilized via WTAP-m6A-IGF2BPs-dependent manner and participated in the regulation of angiogenesis. In addition, Wnt pathway was repressed due to elevated levels of free Wilms' tumour 1 (WT1) in WTAP-deficient endothelial cells, which might be involved in the formation of brain AVMs. Our findings determine the mechanism by which WTAP inhibits angiogenesis and provide potential therapeutic targets to prevent the formation or progression of brain AVMs.

| Patients and samples
Detailed information on patient recruitment and sample preparation could be found in our previous study. 46 Briefly, from September 2016 to November 2017, we recruited 66 patients with brain AVM and seven patients with epilepsy as a control at Beijing Tiantan Hospital affiliated to Capital Medical University. Brain AVMs samples were collected from consecutive patients undergoing surgical treatment.
The clinical diagnoses of brain AVM were confirmed by digital subtraction angiography and histologic evaluation in the hospital's pathology department. In addition, consistent with previous study, 47 intracranial vascular tissue samples without the typical characteristics of brain AVM had been obtained from 7 patients undergoing temporal lobe resection for epilepsy. Informed consents were obtained from all patients, and this study was approved by the institutional review board of Beijing Tiantan Hospital, Capital Medical University.

| Gene silencing and expression
The siRNAs targeting specific genes were designed and synthesized by Guangzhou RiboBio (Table S1). Endothelial cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen). The plasmid expressing Flag-tagged Homo sapiens WTAP was synthesized by Shanghai Genechem Co., Ltd. Constructed plasmid was transfected into the endothelial cells according to the manufacturer's instructions of jetPRIME kit (Polyplus-transfection). After transfection for 48 hours, endothelial cells were harvested for subsequent mRNA or protein expression analysis.

| RNA isolation and qRT-PCR
Total RNAs were extracted and purified using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was reverse transcribed from total RNAs using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa Co). qRT-PCR was performed using the SYBR ® Premix Ex Taq™ II (TaKaRa) on the QuantStudio™ real-time PCR system (Applied Biosystems). Primers for specific genes were listed in Table S2. Finally, the relative analysis of gene expression was evaluated using the 2 −ΔΔC T method.

| Immunofluorescence
The endothelial cells were fixed using 4% paraformaldehyde for 20 minutes. After that, the cells were incubated with 0.3% Triton X-100 for 10 minutes and blocked non-specific binding sites with 5% BSA. Next, the cells were incubated with primary antibodies against m6A (202003; Synaptic Systems) or β-catenin (ab32572, Abcam) overnight at 4°C and subsequently were incubated with Alexa Fluor 594-or Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at room temperature for 1 hour. Finally, nuclear staining was performed with DAPI at room temperature, and the cells were observed using EVOS™ FL Auto 2 Imaging System (Invitrogen).

| Tube formation assay
Tube formation assays were performed using Ibidi μ-Slide Angiogenesis (Ibidi) according to the manufacturer's protocol. A total of 15 000 endothelial cells in 50 μL complete media were plated to the inner well of µ-Slide filled with Matrigel. Then, the μ-Slides were incubated at 37°C as usual. About 24 hours later, the tube formations were imaged under the Fluorescence Inversion Microscope System and analysed using the Image J software.

| RNA stability assays
Endothelial cells were transfected with siRNAs against specific genes or negative control siRNA using Lipofectamine RNAiMAX For m6A-IP-qPCR, the total RNAs were fragmented into 300nt fragments after incubation in fragmentation buffer at 94°C for 30 seconds and immunoprecipitated by anti-m6A antibody according to the procedure shown above. One-tenth of the fragmented RNAs were saved as input control, and the enrichment of m6A was quantified using qRT-PCR.

| Statistical analysis
All experiments were performed at least three independent replicates, and statistical analyses were performed using GraphPad Prism6 software. Statistical significance was calculated by unpaired Student's t test. Results are presented as mean ± SEM, and a P value of less than .05 was considered statistically significant.

| WTAP is down-regulated in brain AVMs lesions and is required for angiogenesis
From September 2016 to November 2017, a total of 66 patients with brain AVM were included in the main study group. Intracranial vascular tissues from seven patients with epilepsy were included as controls. RNA sequencing was performed on collected tissue samples to derive differential gene expression profiles between control and brain AVMs. Through analysing the results of RNA-seq, we identified a total of 5118 genes that were differentially expressed, and that 2680 and 2438 genes were significantly up-regulated and down-regulated in brain AVMs, respectively (Table S3). Surprisingly, we found that although the expression levels of the m6A methyltransferases METTL3 and METTL14 were not significantly different, WTAP was downregulated in brain AVMs lesions compared with normal brain vessels ( Figure 1A; Figure S1; Table S3). In addition, the expression level of WTAP obtained by high-throughput sequencing was confirmed by qRT-PCR ( Figure 1B). To investigate the functional changes in vascular endothelial cells that involved in the pathophysiological process of brain AVMs, we knocked down WTAP in human endothelial cells using specific siRNA ( Figure 1C). The functional assays showed that knockdown of WTAP significantly inhibited the tube formation of vascular endothelial cells ( Figure 1D). Therefore, we concluded that WTAP played a critical role in angiogenesis of vascular endothelial cells.

| Analysis of potential targets for WTAP
Wilms' tumour 1-associating protein has been identified to function as a significant regulatory subunit in the m6A methyltransferase complex and plays a critical role in epitranscriptomic regulation of RNA metabolism. 19 As shown in Figure 2A, silencing of WTAP dramatically reduced the m6A modification level in endothelial cells. To profile the difference in m6A methylation of mRNA, we performed MeRIP-seq using control and WTAP-deficient human endothelial cells. The results showed that the level of m6A modification of 533 genes in WTAP-deficient endothelial cells was significantly lower than that of the control cells ( Figure 2B; Table S4).
Next, we performed RNA sequencing to fully elucidate the molecular mechanisms of m6A function. About 452 up-regulated and 495 down-regulated genes were identified in WTAP-deficient endothelial cells, respectively ( Figure 2C; Table S5). GO enrichment analysis revealed that the up-regulated genes were enriched in tube morphogenesis, regulation of growth and certain metabolic pathways ( Figure 2D). Correspondingly, the down-regulated genes were enriched in immune-related pathway ( Figure 2D). We then analysed the expression levels of 533 WTAP potential targets in WTAP-deficient endothelial cells. About 74 targets were shown to be differentially expressed genes, including six up-regulated and 68 significantly down-regulated genes ( Figure 2E). Therefore, we speculated 74 differentially expressed genes were the directly downstream of WTAP and participated in regulation of tube formation in endothelial cells.
F I G U R E 1 WTAP is down-regulated in brain AVMs and is required for angiogenesis. A, RNA-seq showing the expression levels of WTAP in brain AVMs lesions versus normal vessels. B, Differential expression of WTAP identified in high-throughput sequencing was verified by qRT-PCR. C, qRT-PCR and Western blot analysis of the knockdown efficiency of WTAP in endothelial cells. D, Representative bright-field images and statistical analysis of tube formation assay of control and WTAP-deficient endothelial cells. Data are shown as mean ± SEM of three independent experiments. P values were calculated using Student's t test. *P < .05; **P < .01; ***P < .001

| DSP is the downstream target of WTAP
Previous studies have shown that METTL3 is the most important m6A methyltransferase. Therefore, we also performed MeRIP-seq and RNA-seq on METTLE3 knockdown endothelial cells, and the identified differential expressed genes and differentially methylated peaks had been listed in Tables S6 and S7, respectively. In addition, IGF2BPs have been identified as the m6A readers and promote the stability of their target mRNAs in an m6A-dependent manner. 26,27 In WTAP-deficient endothelial cells, most of the differentially expressed genes in 533 transcripts with lower m6A modification levels were down-regulated. Therefore, we speculated that IGF2BPs as the main m6A readers are involved in the regulation of mRNA stability by WTAP in endothelial cells. Taking these into consideration, we combined the previously published data of IGF2BPs targets identified by RIP and PAR-CLIP with our MeRIP-seq and RNA-seq data on METTL3-and WTAP-deficient endothelial cells for conjoint analysis to further determine the certain target. 26 Since DSP was also the common target for METTL3 and IGF2BPs, it was selected for further analysis among all the potential WTAP targets ( Figure 3A). MeRIP-seq and RNA-seq data showed that knockdown of WTAP or METTL3 sharply reduced enrichment of m6A peaks and significantly down-regulated DSP mRNA levels ( Figure 3B; Table S8) Figure 3G). Surprisingly, we found DSP was significantly down-regulated in brain AVMs lesions compared with normal brain vessels ( Figure 3H; Table S3).
Thus, down-regulated WTAP repressed the expression level of DSP and ultimately inhibited the tube formation of endothelial cells, which may be related to the pathophysiology of brain AVMs.

| The stability of DSP mRNA depends on the m6A reader IGF2BPs
Previous study has demonstrated IGF2BP1/2/3 recognize the consensus GGAC sequence through the K homology domains. 26 We also found that the peak distributed in chr6 7579507-7581802 (HG19) contained the GGAC motif. Subsequently, we knocked down IGF2BP1, IGF2BP2 and IGF2BP3, respectively, to elucidate whether m6A readers IGF2BPs were involved in the regulation of After treatment with actinomycin D, the mRNA levels of DSP were also reduced in IGF2BPs deficient endothelial cells ( Figure 4D-F).
However, RNA stability assays showed that the half-lives of DSP mRNA in IGF2BP1 and IGF2BP3 deficient endothelial cells were dramatically shortened compared with control cells, while the halflife of DSP mRNA in IGF2BP2 deficient cells was slightly changed ( Figure 4D-F). Taken together, these results suggested that methylated DSP mRNAs could be recognized by the IGF2BP1 and IGF2BP3 to prevent degradation and maintain stability.

| WNT signalling pathway is inhibited in WTAPdeficient endothelial cells
It is well known that Wnt signalling plays a central role in embryonic development, differentiation, cell motility, cell proliferation, adult tissue homeostasis, angiogenesis and so on. [50][51][52][53] Furthermore, Wnt signalling can activate canonical β-catenin-dependent pathway and at least two well-characterized β-catenin-independent pathways, the planar cell polarity (PCP) pathway and the Wnt/ Ca2 + pathway. 54,55 However, Wnt signalling pathway has been reported to be inhibited by targeting the WTAP-WT1-TBL1 axis. 56,57 WT1 is a negative regulator of the Wnt signalling pathway.
WTAP can interact with WT1 and form a WTAP/WT1 complex. Therefore, a decrease in WTAP protein levels leads to the release of free WT1, which results in the induction of transducing β-like protein 1 (TBL1) and ultimately reduces the level of β-catenin protein. 56 Therefore, it was worthwhile to explore whether WTAP affects the Wnt signalling pathway through the WT1-TBL1 axis in endothelial cells. Same as previous research results, silencing or overexpression of WTAP did not affect the expression of WT1 in endothelial cells, but reduced or increased the protein level of β-catenin, respectively, without affecting its mRNA levels ( Figure 5A-C). Meanwhile, immunofluorescent staining results indicated that knockdown or overexpression of WTAP inhibited or facilitated the translocation of β-catenin to the nucleus, respectively ( Figure 5D,E). In summary, WTAP could modulate the Wnt signalling pathway via releasing free WT1 in endothelial cells.

| D ISCUSS I ON
Brain AVMs, characterized by direct connection between cerebral arteries and veins, have been identified as the important risk factor in fatal symptoms such as cerebral haemorrhage, epilepsy and stroke. 1,5 But until now, the pathogenesis of brain AVMs is still unclear. In this study, we found WTAP was down-regulated in the lesions of brain AVMs by transcriptome sequencing (Figure 1A, B). And WTAP could affect the angiogenesis of endothelial cells ( Figure 1D). Therefore, down-regulated WTAP might participated in the pathophysiological process of brain AVMs.
Genetic and epigenetic mechanisms have been elucidated to play an important role in the pathogenesis and development of brain AVMs. For example, somatic activating KRAS mutations were detected in most tissue samples of sporadic non-familial brain AVM. 47 In terms of epigenetics, decreased expression of miRNA-18a in endothelial cells cultured from brain AVMs altered the production of anti-and pro-angiogenic factors. 58 Furthermore, aberrant epigenetic modifications in the genome of endothelial cells may drive the artery or vein to an aberrant phenotype.
Previous studies suggested a significant correlation between DNA methylation levels and brain AVM risk. 59 Considering that WTAP is an important regulatory subunit of m6A methyltransferase complex, 19 and can significantly affect the level of m6A modification in endothelial cells (Figure 2A), we speculated that RNA epigenetic modifications-m6A is also likely to be related to angiogenesis and the formation of brain AVM.
In the current study, we performed MeRIP-seq and RNA-seq to reveal underlying molecular mechanisms by which WTAP regulated angiogenesis. Finally, we identified DSP as a downstream target of WTAP-mediated m6A modification ( Figure 3A-F). DSP is a critical component of desmosome and is essential for maintaining the integrity of tissues, especially those under high mechanical stress, such as epidermis and myocardium. 38,40 Moreover, previous studies have demonstrated DSP can modulate gene expression, differentiation and microtubule dynamics, indicating that biological functions for this protein exceed its central role in cell-cell adhesion. 60,61 Particularly, ablation of DSP leads to leaky and/or poorly formed capillaries, limiting embryonic development. 45 Here, we confirmed DSP was an essential component of angiogenesis in endothelial cells ( Figure 3G), and DSP was significantly down-regulated in brain AVMs lesions ( Figure 3H; Table S3). Consequently, WTAP could regulate DSP expression through m6A modification to affect angiogenesis in brain AVMs.
Our findings expanded new developmental processes involving m6A modification in addition to cell differentiation, circadian rhythm, DNA damage response, sex determination, neuronal disorder, infectious diseases and tumorigenesis, and increased our understanding of the pathogenesis of brain AVM. Moreover, there are many other types of modifications in RNA, and their functions are being elucidated. Among them, it is noticeable that m5C, m7G and ac4C have been found to play important regulatory roles in the metabolic process of their modified RNA. [62][63][64] The role of these epigenetic and other epigenetic factors, such as F I G U R E 5 Wnt signalling pathway is inhibited in WTAP-deficient endothelial cells. 56 A, qRT-PCR analysis of the mRNA levels of specific genes in WTAP-deficient or (B) overexpressing endothelial cells. C, Western blot analysis of the protein level of β-catenin after silencing or overexpressing WTAP. D, Immunofluorescent staining for β-catenin in WTAP-deficient or (E) overexpressing endothelial cells. Data are shown as mean ± SEM of three independent experiments. P values were calculated using Student's t test. *P < .05; **P < .01; ***P < .001 histone modifications and chromatin states, in the development of AVM requires further research to improve our understanding of disease.
Wilms' tumour 1-associating protein is a ubiquitously expressed nuclear protein, which was first identified as the partner of WT1, a protein playing an essential role in normal development. 65 WT1 is a transcription factor that governs the expression of a range of effectors, including genes which regulate the Wnt signalling pathway. 66 For instance, degradation of WTAP enhances the WT1-binding activity to induce the expression of TBL1, and this finally results in the degradation of β-catenin. 56 In this study, we further confirmed this conclusion: WTAP can antagonize the activity WT1, which negatively regulates the Wnt signalling pathway ( Figure 5). The Wnt signalling pathway is one of the pivotal regulatory systems in co-ordinating endothelial cells behaviour to govern vascular morphogenesis. [67][68][69] In particular, endothelial-specific loss of β-catenin leads to defective vascular remodelling and impairs the development of the embryonic vasculature. 70 On the basis of the above, we speculated that WTAP could also inhibit angiogenesis by negatively regulating the Wnt pathway.
Overall, our results revealed that WTAP expression level was reduced in lesions of brain AVMs, which would inhibit angiogenesis of endothelial cells. Mechanistically, DSP mRNA was rapidly degraded in WTAP-deficient endothelial cells due to reduced m6A modification. On the other hand, the lack of WTAP enhanced WT1 activity, thereby repressing the Wnt signalling pathway. These findings will contribute to the elucidation of the pathogenesis of brain AVMs and provide potential targets for treatment.

CO N FLI C T O F I NTE R E S T
All authors state that they have no conflicts of interest.